The oligonucleotides of the invention are intended, in general, for application to an approach which has come to be known as "antisense" therapy.
The general principles of antisense therapy are now well recognized. Most diseases and undesirable conditions in humans and animal subjects are mediated by specific DNA or RNA sequences which, if inactivated, would no longer be able to facilitate the progress of the disease. The antisense approach provides DNA or RNA oligomers, or their analogs, which are capable of specific binding to the undesirable nucleic acid sequences. These materials can be supplied directly or generated in situ, and may be conventional oligomers, or are more commonly oligomers having properties which make them, for example, resistant to nucleases or more capable of hybridization to the desired target. The hybridization may be effected by providing oligomers having sequences which result in conventional base-pairing, or these may recognize double-stranded DNA by binding to the major or minor grooves which are present in the double helix. Whatever the ultimate strategy, it is desirable to provide oligomers with physiological properties which render them more effective.
The art provides a number of approaches whereby modified oligonucleotides are used in antisense applications. For example, in order to provide enhanced stability in vivo, through resistance to endogenous nucleases, oligomers have been synthesized with alternative linkages other than the conventional phosphodiester linkage. Among these are the methylphosphonates wherein one of the phosphorous-linked oxygens has been replaced by methyl; phosphorothioates, wherein sulfur replaces one of the oxygens; and various amidates, wherein NH.sub.2 or organic amine derivatives, such as morpholidates or piperazidates, replace an oxygen. Also carbonate and carbamate linkages have been employed, as well as those involving sulfur rather than oxygen as a linking substituent.
In addition, modifications have been employed wherein the oligonucleotides are conjugated with a lipophilic group to enhance cell permeation capability. Inclusion of intercalators and chelators which enhance the ability of the oligonucleotide to bind the target DNA or RNA is also known. These substituents have been attached to the 5' end of preconstructed oligonucleotides using amidite or H-phosphonate chemistry, as described by Ogilvie, K. K., et al., Pure and Appl Chem (1987) 59:325, and by Froehler, B. C., Nucleic Acids Res (1986) 14:5399. Intercalators have also been attached to the 3' end of oligomers, as described by Asseline, U., et al., Tet Lett (1989) 30:2521. This last method utilizes 2,2'-dithioethanol attached to a solid support to displace diisopropylamine from a 3' phosphonate bearing the acridine moiety and is subsequently deleted after oxidation of the phosphorus. Other substituents have been bound to the 3' end of oligomers by alternate methods, including polylysine (Bayard, B., et al., Biochemistry (1986) 25:3730; Lemaitre, M., et al., Nucleosides and Nucleotides (1987) 6:311) and, in addition, disulfides have been used to attach various groups to the 3' terminus, as described by Zuckerman, R., et al., Nucleic Acids Res (1987) 15:5305. It is known that oligonucleotides which are substituted at the 3' end show increased stability and increased resistance to degradation by exonucleases (Lancelot, G., et al., Biochemistry (1985) 24:2521; Asseline, U., et al., Proc Natl Acad Sci USA (1984) 81:3297). A recent report by Mori, K., et al., FEBS Lett (1989) 249:213-218, describes oligonucleotides coupled to anthraquinone at the 5' terminus. The coupled oligomers exhibit anti HIV activity in vitro; and the inclusion of the anthraquinone residue appears to raise the melting temperature. The advantage of this 5' derivatization is said to be the activity of the anthraquinone as an oxidizing agent and to produce radicals.
The general approach to constructing various oligomers useful in antisense therapy has been reviewed by vander Krol, A.R., et al., Biotechniques (1988) 6:958-976, and by Stein, C. A., et al., Cancer Res (1988) 48:2659-2668, both incorporated herein by reference.
The present invention provides oligonucleotides which are coupled to at least one anthraquinone moiety at other than the 5' terminus; the inclusion of this moiety is capable of enhancing the ability of the oligomer to hybridize specifically to target sequences without loss of specificity. In addition, the anthraquinone can serve as a marker and can stabilize the oligomer with respect to nuclease degradation.